PROJECT SUMMARY Cyclic dinucleotides of host and bacterial origin have emerged as ubiquitous second messengers and potent modulators of host immune responses, with important roles in shaping infectious, malignant and autoimmune diseases. The eukaryotic second messenger 2',3'-cGAMP is produced by cGAS in response to DNA within the host cell cytoplasm. In response to DNA derived from bacterial and viral infection cGAMP initiates host inflammation to clear infection, while sensing of self-derived DNA has been implicated in autoimmune disorders including Systemic Lupus Erythemytosus and Aicardi-Goutieres Syndrom. Additionally, bacteria produce a variety of cyclic dinucleotides that function as second messengers and also promote host inflammation during infection. In each of these instances, CDN binding to the mammalian receptor STING promotes inflammatory responses. Despite our current understanding pertaining to CDN mediated inflammation, there is a significant limitation in the capacity to directly measure and observe CDNs within biological settings. To date, CDN detection relies on LC-MS/MS or ELISA based methods. These technologies while important are limited in the spatial and temporal resolution they afford. To overcome these current limitations, we have undertaken the development and validation of a universal, genetically encoded fluorescent CDN biosensor. This sensor relies on the CDN binding domain of STING and affords unparalleled temporal and single cell detection of CDNs in living cells. We now aim to (i) biochemically characterize and establish the in vitro utility of this sensor for monitoring CDN dynamics, (ii) utilize tissue culture studies to validate and characterize the dynamics of CDN levels in living cells, and (iii) apply this new technology to conduct a forward genetic screen for cell intrinsic regulators of cGAS-cGAMP signaling in human cells. Together the studies outlined here will provide an innovative and broadly useful tool to study CDN signaling within eukaryotes and provide potential biological insight into the regulation of the cGAS-cGAMP signaling axis, with important consequences on infectious, malignant, and autoimmune diseases.